The purpose of this project is to investigate epigenetic alterations in CML. We have previously discovered imprinted genes in humans and loss of imprinting (LOI) in cancer. Genomic imprinting is a chromosomal modification in the gamete or zygote leading to differential expression of the two parental alleles of a gene in somatic cells of the offspring. LOI can lead to the expression of the normally silent copy of growth-promoting genes in cancer, and to loss of expression of the normally transcribed copy of growth inhibitory genes. In addition, we have found alterations in DNA methylation, a covalent modification of the nucleotide cytosine, that are specific for tumor cells with LOI. As part of this program project, we have previously discovered LOI in CML, which appears to be specific for progression to accelerated phase and blast crisis. In the current grant period, we have identified specific alterations in methylation of the IGF2 gene that are linked to LOI and are found in circulating leukemic cells of patients with accelerated phase or blast crisis, as well as in the year preceding disease progression. This methylation alteration appears to be specific for leukemic cells, as it is not found in normal circulating myelocytes or marrow progenitor cells. We will now determine whether clinical subtype and response to therapy can be classified by altered methylation of IGF2, and/or by alterations in the epigenotype of a group of genes analyzed by high throughput bisulfite methylation analysis, as well as array-based methylation analysis. These efforts will be used to generate an "epigenetic profile" of CML that can be used for classification and evaluation of therapeutic response as well as therapeutic planning. We will also determine the relationship between clinical subtype or the response to therapy, and histone modifications, as well as the expression of the chromatin modifying genes responsible for these changes. These latter studies will be focused on identifying chromatin factors responsible for Gleevec resistance, as well as for epigenetic silencing of the wild type bcr allele in disease progression, and they will be performed in collaboration with Dr. Talpaz and Dr. Arlinghaus. These experiments will provide direct insight into the pathogenesis of CML progression and help to identify novel targets in the therapy of CML.